By the methods of X-ray protein crystallography, we propose to obtain the structure-function relationship in terms of the three-dimensional structures of various proteins and to establish the evolutionary relationship as expressed in secondary and tertiary structures. In penicillopepsin (pepsin from penicillium zanthinellum) we will attempt to introduce substrate analogs, inhibitors, and activators into the enzyme by soaking or co-crystallization method and to locate these compounds by difference fourier synthesis. Chemical modification and pH dependence study could reveal the structural variations. Some acid proteases have been implicated in cancer spread and heart attack. We also plan to determine the structure of porcine pepsinogen (a zymogen of pepsin) at 2.8 Angstroms by the multiple isomorphous replacement method. The conformational changes upon activation of the zymogen may yield information regarding the catalytic mechanism of acid proteases. With the molecular replacement techniques we propose to solve the structure of porcine intestinal calcium-binding protein using the known parameters of bovine protein. We want to locate the calcium-binding sites, to analyze the interactions of calcium ions with the protein, to delineate the conformational differences between apo- and calcium-bound proteins, and to compare the structure with that of other calcium-binding proteins. Structural studies would aid in understanding the mechanism by which these proteins could function, and the crystallographic results would also yield information on the evolution of these proteins.